ABSTRACT The incidence of esophageal adenocarcinoma (EAC) has increased more than 4-fold in the United States over the past 40 years. Unfortunately, the prognosis for EAC patients is poor with an overall 5-year survival rate of 19%. Due to the lack of effective screening options, EAC patients are often diagnosed late with advanced disease, where the 5-year survival rate is only 0.9%. Current standard-of-care treatments, consisting of neoadjuvant chemotherapy followed by surgical resection are ineffective in the majority of cases. Therefore, a substantial unmet medical need exists to identify and successfully drug not only the drivers of EAC but also the signaling pathways that underlie resistance to chemotherapy. Herein, we present a clear role for Notch signaling in EAC as critical for the maintenance of the neoplastic phenotype conferring resistance to neoadjuvant chemotherapy by driving a cancer stem cell phenotype. The Notch pathway is considered to be a highly attractive and sought after target with no specific pathway inhibitors. Notch signaling involves a series of proteolytic cleavage events that releases the Notch intracellular domain (NICD) from the cell surface. NICD is critical for the formation of an active Notch ternary complex (NTC). The NTC is composed of NICD, CSL, and Mastermind (MAML), without all of these components together the NTC is unable to activate transcription. Herein, we present our novel approach to identify a drug binding pocket in the NTC and then develop a class of small molecule inhibitors that prevent active NTC formation with the aim of complementing and/or replacing existing EAC therapies. We have discovered a novel class of Notch inhibitors and that specifically bind to a pocket in the CSL/NICD interface and prevents binding of MAML and thereby inhibiting Notch signaling. We also present our validation of the mechanism of action and in vivo efficacy of early lead compounds. We also outline the significant progress made in lead optimization that identified analogs with improved potency (>1000-fold) and in the identification of new scaffolds via molecular modeling/scaffold hopping. In this Phase I SBIR application, we propose to evaluate the in vivo efficacy and therapeutic window for the new analogs. Completion of these studies will yield a preclinical candidate for IND-enabling studies. StemSynergy Therapeutics, Inc (SSTI) is a biopharmaceutical company whose focus is on the discovery, development, and commercialization of drugs targeting WNT, Hedgehog and Notch signaling pathways- pathways critical to stem cell and cancer stem cells. The Notch inhibitors identified in this proposal, 1- 134-83 and analogs, represent new chemical entities and a patent application has been filed. SSTI has an exclusive license to develop this compound series and the expertise and the opportunity to bring to market a first-generation Notch inhibitor that would capture a significant share of the global market for cancer drugs, not just to treat EAC, but also for other Notch-driven cancers.